The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become an extremely useful tool for tracking and quantifying biological entities in the fields of biochemistry, molecular and cell biology, and medical diagnostics (Chalfie et al., 1994, Science 263:802–805; Tsien, 1998, Ann. Rev. Biochem. 67:509–544). There are no cofactors or substrates required for fluorescence, thus the protein can be used in a wide variety of organisms and cell types. GFP has been used as a reporter gene to study gene expression in vivo by insertion downstream of a test promoter. The protein has also been used to study the subcellular localization of a number of proteins by direct fusion of the test protein to GFP, and GFP has become the reporter of choice for monitoring the infection efficiency of viral vectors both in cell culture and in animals. In addition, a number of genetic modifications have been made to GFP resulting in variants for which spectral shifts correspond to changes in the cellular environment such as pH, ion flux, and the phosphorylation state of the cell. Perhaps the most promising role for GFP as a cellular indicator is its application to fluorescence resonance energy transfer (FRET) technology. FRET occurs with fluorophores for which the emission spectrum of one overlaps with the excitation spectrum of the second. When the fluorophores are brought into close proximity, excitation of the “donor” fluorophore results in emission from the “acceptor”. Pairs of such fluorophores are thus useful for monitoring molecular interactions. Fluorescent proteins such as GFP are useful for analysis of protein:protein interactions in vivo or in vitro if their fluorescent emission and excitation spectra overlap to allow FRET. The donor and acceptor fluorescent proteins may be produced as fusions with the proteins one wishes to analyze for interactions. These types of applications of GFPs are particularly appealing for high throughput analyses, since the readout is direct and independent of subcellular localization.
Purified A. victoria GFP is a monomeric protein of about 27 kDa that absorbs blue light with excitation wavelength maximum of 395 nm, with a minor peak at 470 nm, and emits green fluorescence with an emission wavelength of about 510 nm and a minor peak near 540 nm (Ward et al., 1979, Photochem. Photobiol. Rev. 4:1–57). The excitation maximum of A. victoria GFP is not within the range of wavelengths of standard fluorescein detection optics. Further, the breadth of the excitation and emission spectra of the A. victoria GFP are not well suited for use in applications involving FRET. In order to be useful in FRET applications, the excitation and emission spectra of the fluorophores are preferably tall and narrow, rather than low and broad. There is a need in the art for GFP proteins that are amenable to the use of standard fluorescein excitation and detection optics. There is also a need in the art for GFP proteins with narrow, preferably non-overlapping spectral peaks.
The use of A. victoria GFP as a reporter for gene expression studies, while very popular, is hindered by relatively low quantum yield (the brightness of a fluorophore is determined as the product of the extinction coefficient and the fluorescence quantum yield). Generally, the A. victoria GFP coding sequences must be linked to a strong promoter, such as the CMV promoter or strong exogenous regulators such as the tetracycline transactivator system, in order to produce readily detectable signal. This makes it difficult to use GFP as a reporter for examining the activity of native promoters responsive to endogenous regulators. Higher intensity would obviously also increase the sensitivity of other applications of GFP technology. There is a need in the art for GFP proteins with higher quantum yield.
Another disadvantage of A. victoria GFP involves fluctuations in its spectral characteristics with changes in pH. At high pH (pH 11–12), the wild-type A. victoria GFP loses absorbance and excitation amplitude at 395 nm and gains amplitude at 470 nm (Ward et al., 1982, Photochem. Photobiol. 35:803–808). A. victoria fluorescence is also quenched at acid pH, with a pKa around 4.5. There is a need in the art for GFPs exhibiting fluorescence that is less sensitive to pH fluctuations.
Further, in order to be more useful in a broad range of applications, there is a need in the art for GFP proteins exhibiting increased stability of fluorescence characteristics relative to A. victoria GFP, with regard to organic solvents, detergents and proteases often used in biological studies. There is also a need in the art for GFP proteins that are more likely to be soluble in a wider range of cell types and less likely to interfere non-specifically with endogenous proteins.
A number of modifications to A. victoria GFP have been made with the aim of enhancing the usefulness of the protein. For example, modifications aimed at enhancing the brightness of the fluorescence emissions or the spectral characteristics of either the excitation or emission spectra or both have been made. It is noted that the stated aim of several of these modification approaches was to make an A. victoria GFP that is more similar to R. reniformis GFP in its excitation and emission spectra and fluorescence intensity.
Literature references relating to A. victoria mutants exhibiting altered fluorescence characteristics include, for example, the following. Heim et al. (1995, Nature 373:663–664) relates to mutations at S65 of A. victoria that enhance fluorescence intensity of the polypeptide. The S65T mutation to the A. victoria GFP is said to “ameliorate its main problems and bring its spectra much closer to that of Renilla”. 
A review by Chalfie (1995, Photochem. Photobiol. 62:651–656) notes that an S65T mutant of A. victoria, the most intensely fluorescent mutant of A. victoria known at the time, is not as intense as the R. reniformis GFP.
Further references relating to A. victoria mutants include, for example, Ehrig et al., 1995, FEBS Lett. 367:163–166); Surpin et al., 1987, Photochem. Photobiol. 45(Suppl):95S; Delagrave et al., 1995, BioTechnology 13:151–154; and Yang et al., 1996, Gene 173:19–23.
Patent and patent application references relating to A. victoria GFP and mutants thereof include the following. U.S. Pat. No. 5,874,304 discloses A. victoria GFP mutants said to alter spectral characteristics and fluorescence intensity of the polypeptide. U.S. Pat. No. 5,968,738 discloses A. victoria GFP mutants said to have altered spectral characteristics. One mutation, V163A, is said to result in increased fluorescence intensity. U.S. Pat. No. 5,804,387 discloses A. victoria mutants said to have increased fluorescence intensity, particularly in response to excitation with 488 nm laser light. U.S. Pat. No. 5,625,048 discloses A. victoria mutants said to have altered spectral characteristics as well as several mutants said to have increased fluorescence intensity. Related U.S. Pat. No. 5,777,079 discloses further combinations of mutations said to provide A. victoria GFP polypeptides with increased fluorescence intensity. International Patent Application (PCT) No. WO 98/21355 discloses A. victoria GFP mutants said to have increased fluorescence intensity, as do WO 97/20078, WO 97/42320 and WO 97/11094. PCT Application No. WO 98/06737 discloses mutants said to have altered spectral characteristics, several of which are said to have increased fluorescence intensity.
In addition to A. victoria, GFPs and other fluorescent proteins have been identified in a variety of other coelenterates and anthazoa. Other GFPs cloned include A. victoria (Prasher, 1992, Gene 111:229–233) and Renilla mulleri (WO 99/49019). A red-shifted fluorescent protein cloned from the coral Discosoma (Matz, M. V. et al., 1999, Nat. Biotechnol. 17:969–973) and named DsRed. Biochemical properties of DsRed and a mutant of DsRed are reported in Baird, G. S. et al. (2000, Proc. Natl. Acad. Sci. USA 97:11984–89).
The native R. reniformis protein was purified and characterized by Ward, W. et al. (J. Biol. Chem. 254 3:781–788) in 1979. The native protein was found to have a 5 fold greater extinction coefficient than native A. victoria GFP. In addition the R. reniformis GFP forms a non-dissociable homodimer, shows a broad range of pH stability, is resistant to organic solvents, detergents, and proteases, and has a narrow excitation and emission spectra. RT-PCR with gene specific primers has revealed the presence of two distinct isoforms of R. reniformis GFP (WO 01/68824) and (WO 01/64843).